We are interested in the transcriptional control of T cell development and function. Over the past few years, most of our effort has been dedicated to elucidating the gene expression programs that control the choice by intrathymic T cell precursors of the CD4 or CD8 lineage, and determine their distinctive functional responses. T cells are essential for immune responses. 'Conventional' T cells recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins (called coreceptors) that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and is essentially dictated by MHC specificity. That is, the general rule is that MHC I-specific T cells are CD4-CD8+ (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- (CD4 cells). In addition, CD4 and CD8 T cells typically perform distinct functions upon antigen encounter: whereas CD8 T cells differentiate into cytotoxic effectors, CD4 T cells provide help to other components of the immune system (and of mucosal barriers) and have essential regulatory functions. Both the divergence of CD4 and CD8 lineages, and their pre-programming for helper and cytotoxic functions, respectively, occur in the thymus. The development of CD4 T cells, which, because of their central role in immune responses, are essential to control infections (they are the key target of the human immunodeficiency virus HIV), has remained a key area of focus in the laboratory. Our previous studies had shown that the transcription factor Thpok is required for CD4 T cell differentiation in the thymus and notably represses CD8-lineage gene expression and CD8 T cell differentiation. We are currently pursuing investigations of the transcriptional circuitry driving CD4-lineage differentiation in the thymus by interrogating the functional relationships between Thpok and other factors involved in this process, including Gata3, Tox and E-box binding proteins E2A and HEB. Furthermore, we had shown that the combined activities of Thpok and the related transcription factor LRF (Leukemia-Lymphoma Related Factor) are essential for the emergence of helper effector functions in MHC II-restricted thymocytes. More recently, we examined the role of Thpok and LRF in post-thymic (mature) CD4 T cells. Both factors are expressed in essentially all mature CD4 T cells, as determined by flow cytometric analyses of intra-cellular protein expression or in 'reporter' mouse strains. Using genetic approaches to inactivate the genes encoding Thpok and LRF in post-thymic T cells, we recently reported that the combined activity of both factors is required to maintain the 'integrity' and function of CD4 T cells. Disruption of these factors alters the functional responses of CD4 T cells, as assessed by their cytokine production or expression of surface molecules needed for helper functions. Current studies investigate the mechanistic bases of these critical functions of Thpok and LRF. While CD4+ T cell differentiation remains a key focus of the laboratory, we also explored the functions of a specific epigenetic mark, tri-methylation of histone H3 lysine 27 (H3K27Me3), in T cell development. In addition to transcription factors, chromatin structure and covalent DNA or histone modifications are essential for proper control of gene expression. Particular attention has focused on histone modifications, including methylation of specific lysine residues. Notably, trimethylation of histone H3 lysine 27 at gene promoters is associated with transcriptional repression. In studies initiated in collaboration with John O'Shea's laboratory (NIAMS), we had found that the Thpok locus was highly 'decorated' with H3K27Me3 in DP or CD8 SP thymocytes, which do not express Thpok, whereas the mark was entirely removed in CD4 SP thymocytes, which express Thpok. This observation suggested that H3K27Me3 removal was associated with induction of Thpok expression, prompting us to investigate the control of H3K27Me3 homeostasis in developing T cells. H3K27 is methylated by Ezh1 and Ezh2, the latter being the most important in T cells. The tri-methylated form, H3K27Me3, recruits Polycomb Regulatory Complex 1, whose enzymatic activities contribute to transcriptional repression. H3K27Me3 can be demethylated by Utx and Jmjd3, two enzymes which are part of a large family of histone demethylases sharing a similar JmjC catalytic domain. Unlike other developmental processes, CD4-differentiating thymocytes are non-dividing, implying that H3K27Me3 removal does not result from 'dilution' during cell proliferation. Our observation that H3K27Me3 is removed from the Thpok promoter in differentiating CD4 SP thymocytes led us to speculate that H3K27Me3 demethylation by Jmjd3 and Utx would be important for proper gene expression in these cells. To test this hypothesis, we generated mice with T cell specific-disruption of Jmjd3 and Utx. Analyses of these animals showed that these enzymes are required for T cell differentiation, and for H3K27Me3 removal at, and expression of genes involved in thymocyte maturation. In addition, we found that Jmjd3 and Utx are required for the differentiation of iNK T cells, a subset of 'innate-like' lymphocytes that recognize CD1d-bound lipid molecules. Collaborative work in the laboratory of Dr Alexander Tarakhovsky (Rockefeller University) found evidence that H3K27 methylation-demethylation contributes to controlling the promoter of the gene encoding PLZF, a transcription factor required for iNK T cell development. These studies identify critical but unexpected functions of H3K27Me3 demethylases Jmjd3 and Utx in CD4+ T cell development, and provide evidence for both demethylase-dependent and -independent functions of these enzymes. They further demonstrate that these enzymes are required for H3K27Me3 homeostasis, although their highly specific impact, even in non-dividing cells, suggests that alternative mechanisms contribute to this process.